The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels. To this end we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE and mutations in which cause severe blindness in humans. Disruption of the RPE-based vitamin A visual cycle metabolism of all-trans-retinyl esters to 11-cis-retinal appears to underlie the phenotype of the Rpe65 knockout mouse. The function of RPE65 thus appears to be associated with that of the retinol isomerase, the crucial enzyme in visual pigment regeneration. We have also continued studies on beta-carotene 15,15'-monooxygenase (beta-CM). Beta-CM is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that beta-CM and RPE65 may share a similar mechanism of action. In the past year we have made the following progress: a) The role of residues conserved in all members of the carotenoid-cleavage enzyme family (including beta-CM and RPE65) has been investigated by site-directed mutagenesis of putative metal binding residues in beta-CM. The data show a crucial role in enzymatic activity for residues hypothesized to be involved in metal coordination. Mutation of the different residues results in loss of iron-binding and/or changes in Km and Vmax of mutant proteins. b) RPE65 contains a caveolin-interaction domain that is conserved in all species and is functionally active. Caveolin-1 is a "scaffolding" protein known to be involved in membrane trafficking, lipid transport and in signal transduction. Its interaction with RPE65 may involve some or all of these roles. We have found that RPE65 binds to both the scaffolding domain and the C-terminal domain of caveolin-1. This interaction, along with the previously shown interaction with phospholipids, may allow for the association of RPE65 with membrane anchored visual cycle components and provides a new focus for study of the visual cycle complex. c) Analysis of the function of the beta-CM promoter demonstrates that it is regulated by a peroxisome proliferator activated receptor (PPAR) response element (PPRE). Transient transfection of beta-CM promoter-reporter constructs into monkey RPE, ARPE19 and the TC7 and PF11 subclones of the Caco-2 human enterocytes cell line demonstrated that a consensus PPRE is required for optimal activation of the gene. Electrophoretic mobility shift experiments showed that nuclear proteins from monkey RPE cells and TC7 and cell line bind to the PPRE element, while supershift experiments with antibodies to PPARs demonstrated the binding of PPARgamma to the PPRE site in this promoter. Furthermore, we showed, by co-transfection with PPARgamma and RXRalpha and activation with PPARgamma agonists LY 17883 and Ciglitazone, that the PPRE element confers peroxisome proliferator responsiveness via the PPARgamma and retinoid X receptor (RXR) alpha heterodimer. d) The identity of putative factors binding to transcription elements in the RPE65 gene promoter is being sought. Expression clones for these factors are being tested for their effect on activation of the RPE65 promoter. e) Collaboration continues on the rescue of the Rpe65 knockout mouse phenotype and the Briard dog RPE65 dystrophy by AAV-mediated gene transfer. An increased sensitivity of electrophysiological and behavioral responses to light in treated mice and dogs has been noted. The significance of uveitis in some treated dogs has been investigated.